Fig. 7.

BHPI-induced necrosis is an upstream effect of anticipatory UPR activation and requires ATP depletion. a Incorporation of ³⁵S-methionine into newly synthesized protein in TYS cells treated for 1 h with vehicle (set to 100%), 50 nM BHPI, 200 nM ISRIB, BHPI + ISRIB, or 10 µM cyclohexamide (CHX). b ISRIB does not prevent BHPI-induced cell death. Trypan blue exclusion assay for cell death in TYS cells after 1 h treatment with vehicle, 1 µM BHPI, 200 nM ISRIB, or BHPI + ISRIB. c Locking the EnR IP₃R calcium channels closed with 2-APB blocks BHPI-induced cell death. Trypan blue exclusion assay for cell death in MCF-7 and TYS cells treated for 1 h with vehicle, 1 µM BHPI, 100 µM 2-APB, or BHPI + 2-APB. d THG inhibits BHPI-induced cell death. Trypan blue exclusion assay for cell death in TYS and MCF-7 cells treated for 1 h with vehicle, 1 µM BHPI, 10 µM THG, or BHPI + THG. e Time-dependent decline in ATP levels in BHPI-treated cells correlates with cell death. Measurement of whole-cell ATP levels (bars) and trypan blue exclusion (overlay, [ο]) after treatment for the indicated times with vehicle, 1 µM BHPI, 10 µM THG, or BHPI + THG. For TYS cells, ~23% of cells have died at early times and disintegrated at 24 h and are not counted by the instrument. Therefore, the percentage of trypan blue excluding cells declines at 24 h, the total number of dead cells (trypan blue positive + disintegrated cells) is estimated to be ~59%. Data are mean ± s.e.m. a (n = 4 biological replicate experiments), b−d (n = 3 biological replicate experiments), e (n = 5 biological replicate experiments; ATP) (n = 3 biological replicate experiments; cell viability) *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant by Student’s t test